Carbohydrates such as glucose and mannose are ionizable to anions at high pH and can therefore be separated on anion exchange chromatography columns in sodium hydroxide eluents.
Known anion-exchange compositions generally fall into several categories. In the more traditional anion-exchange systems, synthetic support resin particles, generally carrying a negative charge, are covered with a layer of smaller synthetic resin particles carrying anion-exchange functional groups of positive charge, i.e. anion-exchange sites. The smaller particles are retained on the larger support particles via electrostatic attraction. The support resin can take a variety of forms. See for example U.S. Pat. Nos. 4,101,460; 4,383,047; 4,252,644; 4,351,909; and 4,101,460.
A more recent development utilizes an uncharged support resin and smaller latex particles containing anion-exchange functional groups, held together by a dispersant. See U.S. Pat. No. 5,324,752.
In addition, methods have been developed to eliminate the smaller latex particles altogether. For example, an anion exchange functionality is grafted, or covalently bonded, to a variety of polymeric substrates; see for example U.S. Pat. No. 5,006,784. Alternatively, the anion-exchange functional groups are not covalently attached but are tightly associated with the support resin particles, either electrostatically or otherwise; see U.S. Pat. No. 4,927,539.
Most carbohydrates, which are neutral under normal conditions and thus are not retained by anion exchange, can be retained and separated if the pH of the stationary phase is high enough. Eluent pH in the range of 12 to 14 is necessary to insure that carbohydrates are at least partially ionized, based on their dissociation constants. The pH of the stationary phase is a function of the concentration of hydroxide. Traditionally, the concentration of hydroxide in the stationary phase is adjusted by one of four methods, all of which have significant drawbacks:
1. Eluent composition: For carbohydrate chromatography, eluents-normally contain hydroxide. A hydroxide-only eluent system will provide the highest stationary phase pH possible. Addition of a secondary anion such as acetate to the eluent will result in a decrease in the stationary phase pH.
2. Crosslink of the stationary phase: Since crosslink controls the water content of the stationary phase by directly controlling the extent of swelling of the stationary phase in water, it also controls stationary phase pH when using a hydroxide-based eluent system. The lower the water content of the stationary phase, the lower the "dilution" of the stationary phase functional groups with water. This is turn results in an increase in the stationary phase pH. The limitation to this method of raising pH is that mass transport in the stationary phase is slowed by raising the crosslink much above the 5% level. This, therefore, represents an upper boundary to the stationary phase pH based on crosslink control. Further increases in crosslink will adversely effect chromatographic performance.
3. Size of functional group: The size of the functional group at the ion exchange site is to a modest extent capable of affecting the stationary phase pH when using a hydroxide-based eluent system. Changing the absolute size of the functional group allows minor adjustments in stationary phase pH by virtue of a diluent effect. As the size of the functional group is increased the total stationary phase volume is "diluted" by the larger volume occupied by the functional group. This approach has two problems. First, in order to have a significant effect on stationary phase pH, the mass of the functional group must be large in comparison to that of the monomer used to create the stationary phase. Under these conditions, there is generally a problem with stationary phase mass transport due to the steric effects of this large functional group. Second, this approach is only useful for reducing the stationary phase pH since the smallest possible functional group (i.e. the quaternary ion exchange site derived from the reaction of vinylbenzylchloride (VBC) and trimethylamine) is commonly used in the preparation of ion exchange sites. Thus the only option in this control mechanism is to increase the size of the functional group which has the effect of reducing the stationary phase pH.
4. Functional monomer fraction: Variation of the functional monomer fraction of the total stationary phase polymer mass can be used to control the stationary phase pH. For example an increase in the fraction of VBC in a latex particle will lead to a higher stationary phase pH when using a hydroxide-based eluent system. This, however, can only be accomplished by decreasing the fractional content of some other monomer in the latex. Typically the content of the latex is already 95% VBC and thus further increases in VBC content of the latex would have only a marginal impact on the stationary phase pH. Furthermore, the remainder of the monomer fraction in the typical latex particle used for high pH anion exchange chromatography of carbohydrates is the crosslinking monomer divinylbenzene (DVB). As mentioned above, lowering the crosslink level in the latex particle in order to allow an increase in the VBC content of the latex would actually have the opposite effect on the stationary phase pH. The increased swelling due to the lower DVB content would more than offset the minor increase due to the slightly higher monomer fraction of VBC. Thus, while variation of the functional monomer fraction may be a useful method of stationary phase pH control, current polymer formulations already provide the maximum stationary phase pH possible with this control mechanism.
Accordingly, it is an object of the present invention to provide compositions for use in ion exchange chromatography that can increase the effective stationary phase pH and thus improve the separation of a wide variety of carbohydrates.
It is a further object to provide methods for making such compositions, and for methods of using the compositions in the separation of carbohydrates.